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446
CASE REPORT
Endoglin germline mutation in a patient with hereditary
haemorrhagic telangiectasia and dexfenfluramine
associated pulmonary arterial hypertension
A Chaouat, F Coulet, C Favre, G Simonneau, E Weitzenblum, F Soubrier, M Humbert
...............................................................................................................................
Thorax 2004;59:446–448. doi: 10.1136/thx.2003.11890
Dexfenfluramine associated pulmonary arterial hypertension
occurring in a patient with hereditary haemorrhagic
telangiectasia related to a mutation within the endoglin gene
is described. This report highlights the critical role of the TGFb signalling pathway in this condition.
P
ulmonary arterial hypertension (PAH) is defined as a
group of diseases characterised by a progressive increase
in pulmonary vascular resistance leading to right heart
failure and ultimately to death.1 Familial cases segregate as
an autosomal dominant trait with low disease gene penetrance. Mutations within the bone morphogenetic protein
receptor type II gene (BMPR2), coding for a receptor of the
transforming growth factor b (TGF-b) superfamily, have
been shown to underlie most familial cases of primary
pulmonary hypertension2 3 and at least 26% of so-called
‘‘sporadic’’ cases.4
Pulmonary arterial hypertension can also develop in
patients with various associated conditions including hereditary haemorrhagic telangiectasia5 or exposure to anorectic
agents such as fenfluramine derivatives.6 The frequency of
PAH is evaluated at one in 10 000 to one in 100 000 patients
with a history of fenfluramine derivatives intake.7
Conversely, since BMPR2 mutations have been detected in
9% of patients with fenfluramine or dexfenfluramine
associated PAH, associated environmental, epigenetic or
genetic factors are required for the development of PAH.7
Mutations in two genes encoding TGF-b receptors (activin
receptor-like kinase 1 (ALK1) and endoglin) underlie
hereditary haemorrhagic telangiectasia, an autosomal dominant vascular dysplasia with abnormally dilated vessels
forming mucosal and visceral telangiectasia.8 Approximately
25% of families affected in France and in the UK carry a
known endoglin germline mutation. Trembath and colleagues5 recently described cases of PAH in families affected
by hereditary haemorrhagic telangiectasia and mutations
of ALK1 were identified in these subjects.
We describe a case of dexfenfluramine associated PAH
occurring in a patient with hereditary haemorrhagic telangiectasia related to a mutation within the endoglin gene. This
report highlights the critical role of the TGF-b signalling
pathway in this condition.
CASE REPORT
Clinical findings
A 34 year old woman was referred with a 3 year history of
progressive dyspnoea on exertion. She had recurrent epistaxes and mucocutaneous telangiectasia and both symptoms
were also observed in three first degree and five second
www.thoraxjnl.com
degree relatives (fig 1), establishing the diagnosis of
hereditary haemorrhagic telangiectasia. She had taken
dexfenfluramine for a period of 10 months 3 years before
the onset of symptoms. On admission, dyspnoea was present
for mild exercise (functional class III of the New York Heart
Association classification). Apart from signs of hereditary
haemorrhagic telangiectasia, physical examination revealed a
systolic murmur of tricuspid regurgitation. A diagnosis of
PAH was established using the diagnostic strategy recommended by the report of the 1998 World Symposium on
Primary Pulmonary Hypertension (fig 2). No evidence of
hepatic or pulmonary arteriovenous malformation was
found. Right heart catheterisation confirmed severe PAH
with a mean pulmonary artery pressure of 46 mm Hg, a
capillary wedge pressure of 5 mm Hg, and a cardiac index of
2.2 l/min6m2. There was no acute response to vasodilators
and the patient was subsequently treated with continuous
intravenous epoprostenol.
Genetic studies
Written informed consent for the genetic diagnosis was
obtained from the patient, according to bioethical laws.
DNA was isolated from peripheral blood lymphocytes using
a purification kit (QIAamp blood kit, Qiagen, Courtaboeuf,
France). All coding exons of the endoglin, ALK1 and BMPR2
genes were sequenced by PCR amplification with intronic
flanking primers and additional exonic primers when
necessary. Primers were designed according to published
sequences9 10 and using the human genome sequence.
Sequencing reactions were performed with the ABI Prism
Big Dye terminator cycle sequencing kit and analysed on an
ABI310 sequence analyser (both from Applied Biosystems,
Foster City, CA, USA).
A single nucleotide deletion of guanine in the coding
region of the endoglin gene was found 470 bases from the
translation start site in exon 11 (del 470G, fig 3). This
alteration results in a frame shift mutation responsible for
premature termination of the protein at position 490. The
truncated protein is predicted to lose at least the cytoplasm
tail and the transmembrane region.
DISCUSSION
We describe dexfenfluramine associated PAH in a patient
with hereditary haemorrhagic telangiectasia probably
favoured by a mutation within the endoglin gene. A germline
endoglin mutation was identified in this case, showing that
both endoglin and ALK1 can be involved in PAH and
hereditary haemorrhagic telangiectasia. This provides confirmation that several components of the TGF-b signalling
pathway are involved in the pathophysiology of PAH.
Moreover, since pulmonary hypertension occurred after a
10 month exposure to dexfenfluramine, it is likely that
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Endoglin germline mutation
447
I
II
III
Risk factors, symptoms and
83
physical examination
Chest radiography
69
36
65
54
59
63
51
ECG
48
TT echo
Strong evidence of PH
34
38
Exclude PH associated with
respiratory disease and/or
IV
hypoxaemia
15
7
4
Figure 1 Pedigree of the family with coexisting pulmonary arterial
hypertension and hereditary haemorrhagic telangiectasia. Squares
denote male family members, circles female family members, hatched
symbol member with both pulmonary arterial hypertension and
hereditary haemorrhagic hypertension (arrow), black symbols members
with hereditary haemorrhagic telangiectasia alone, and open symbols
members unaffected by either condition.
Exclude CTEPH
Ventilation-perfusion
Search for associated
diseases
lung scan
Blood tests including
Pulmonary function tests
If any subsegmental
antinuclear antibody, HIV
ABGs
or segmental defect
and liver function tests
HRCT
Spiral CT and
In some patients TE echo
Nocturnal oxymetry
eventually pulmonary
with echo-enhancing
artery angiography
agent
Confirm PAH and assess
severity
Right heart catheterisation
and acute vasodilator test
exposure to appetite suppressants and endoglin mutation
played a combined role in the development of the pulmonary
vascular disease.6 7 Previous descriptions of hereditary haemorrhagic telangiectasia with PAH and the rare occurrence of
pulmonary hypertension after exposure to dexfenfluramine
suggest that the possibility of a fortuitous association is
unlikely. The frequency of such an association calculated
from the product of the estimated frequency of each disease
would be between 1029 and 1028.7 8
BMPR2, ALK1 and endoglin belong to the TGF-b superfamily
signalling pathway. The TGF-b signalling pathway has
several functions including embryonic angiogenesis, regulation of cell proliferation and differentiation, and extracellular
matrix deposition. It consists of a cascade of molecule
phosphorylation after binding of the ligand at the cell
membrane. After ligand binding a type II receptor phosphorylates and aggregates with a type I receptor and, in some cells
(including endothelial cells and vascular smooth muscle
cells) with endoglin (also called a type III receptor).11 These
receptors have serine/threonine kinase activity and phosphorylate Smad proteins which, in cooperation with coSmads, penetrate the nucleus and activate or inhibit the
transcription of target genes.11 The complexity and versatility
of the TGF-b pathway comes from the number of molecules
belonging to each class in the pathway, which differs from
cell to cell.
Heterozygous mutations are responsible for a 50% decrease
in the level of expression of the disease gene in target cells
such as endothelial or vascular smooth muscle cells, and this
was observed in cells and tissues of patients bearing endoglin
mutations.12 These latter data and the in vitro expression
analysis of endoglin mutants suggest that haploinsufficiency is
the responsible mechanism in most cases. ALK1 or endoglin
haploinsufficiency is responsible for hereditary haemorrhagic
telangiectasia with high penetrance, in contrast to the rare
occurrence of PAH in patients with ALK1 mutations and to
the low penetrance of BMPR2 mutations for PAH. It is
therefore speculated that additional factors are required to
explain the occurrence of a secondary disease with
abnormal proliferation of vascular smooth muscle cells and
endothelial cells in pulmonary arterial lesions. In the case
reported here the additional factor could be exposure to
dexfenfluramine.
TGF-b has an inhibitory role in cell proliferation, particularly on vascular smooth muscle cells. Two growth abnormalities have been described for pulmonary artery smooth
muscle cell in vitro in patients with primary pulmonary
hypertension. The first is a paradoxical proliferative effect of
Figure 2 Diagnostic strategy in patients with suspected pulmonary
arterial hypertension (PAH). ECG = electrocardiogram;
TT = transthoracic; PH = pulmonary hypertension; ABGs = arterial blood
gases, HRCT = high resolution thoracic computed tomography,
TE = transoesophageal.
Figure 3 Sequencing of exon 11 of the endoglin gene. A single
nucleotide heterozygous deletion in the coding region 470 bases from
the translation start site in exon 11 was found. This deletion predicts a
frameshift and a premature stop codon at position 490. The location of
the guanine deletion (del 470G) is indicated by a vertical arrow. A and B
templates correspond to the sequence of an individual without the
mutation and the sequence of the proband’s exon 11, respectively.
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448
TGF-b in pulmonary artery smooth muscle cells from patients
with primary pulmonary hypertension in contrast to control
cells in which TGF-b has a growth inhibitory effect.13 The
second is an excessive uptake of serotonin in pulmonary
artery smooth muscle cells from patients with primary
pulmonary hypertension compared with controls.14 The
vascular smooth muscle cells from patients with primary
pulmonary hypertension also exhibit increased thymidine
incorporation in response to serotonin, a known proliferative
agent for these cells (an effect abolished by serotonin
transporter inhibitors such as fluoxetine) and associated
with increased expression of serotonin transporters.14
Fenfluramine-like medications contribute to the development of PAH by increasing circulating serotonin levels. They
may also act as serotonin transporter substrates to produce
the same effect as serotonin, or they may alter serotonin
transporter expression. An alternative hypothesis could be
related to the inhibitory effect of dexfenfluramine on voltage
gated potassium channels which is responsible for an
increase in the intracellular calcium concentration.15 A
chronic increase in the intracellular calcium concentration
may subsequently promote cell proliferation. In our patient
exposure to dexfenfluramine may have triggered the pulmonary vascular disease.
Under normal conditions the balance between inhibitory
and stimulatory cell growth systems of vascular cells is
maintained by antagonistic factors such as TGF-b, on one
hand, and serotonin on the other. Disturbance of the
equilibrium can originate in mutations of BMPR2, ALK1
or—as suggested by the case presented here—in mutations of
endoglin. Our results are consistent with those of Du and
colleagues16 who showed that the TGF-b cell signalling
pathway could be involved in all forms of pulmonary
hypertension. The finding in our case adds endoglin mutations
to other confirmed or suspected genetic predispositions to
PAH. It also suggests that all environmental risk factors for
PAH must be avoided in patients with hereditary haemorrhagic telangiectasia.
ACKNOWLEDGEMENTS
The authors thank Dr B Thomas for her critical reading of the
manuscript.
.....................
Authors’ affiliations
A Chaouat, C Favre, E Weitzenblum, Service de Pneumologie, Hôpital
de Hautepierre, Strasbourg, France
F Coulet, F Soubrier, Laboratoire de Génétique Moléculaire, Hôpital
Tenon, Assistance Publique-Hôpitaux de Paris, France
G Simonneau, M Humbert, UPRES EA2705, Service de Pneumologie et
Réanimation Respiratoire, Hôpital Antoine-Béclère, Université Paris-Sud,
Assistance Publique-Hôpitaux de Paris, Clamart, France
www.thoraxjnl.com
Chaouat, Coulet, Favre, et al
Correspondence to: Dr A Chaouat, Service de Pneumologie, Hôpital de
Hautepierre, Avenue Molière, 67098 Strasbourg Cedex, France;
[email protected]
This study was supported by grants from Legs Poix, Université Paris-Sud,
AFM and INSERM.
Received 19 June 2003
Accepted 14 August 2003
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Endoglin germline mutation in a patient with
hereditary haemorrhagic telangiectasia and
dexfenfluramine associated pulmonary arterial
hypertension
A Chaouat, F Coulet, C Favre, G Simonneau, E Weitzenblum, F Soubrier
and M Humbert
Thorax 2004 59: 446-448
doi: 10.1136/thx.2003.11890
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